Plasticity is an important feature of neural systems, including the neural system controlling breathing. Despite its potential biological and clinical significance, our understanding of mechanisms giving rise to any form of respiratory plasticity is incomplete. In this revised application for competitive renewal, investigations will be continued concerning cellular mechanisms giving rise to phrenic long-term facilitation, an important model of respiratory plasticity induced by acute exposure to intermittent hypoxia. In a rat model mechanisms of long- term facilitation will be investigated using multiple experimental approaches, including nerve recordings in anesthetized rats, measurements of ventilation in unanesthetized rats, application of RNA interference, a novel method of regulating potentially important genes that play a role in respiratory plasticity, protein analysis and fluorescent methods in fixed tissues to determine expression changes in important molecules, such as reactive oxygen species. Using these approaches, five hypotheses will be tested in this application for competitive renewal. First, in Aim 1, the hypothesis that the proteins NADPH oxidase and protein phosphatase 2A function as key regulators of long term facilitation will be explored. Next, the possibility that distinct cellular mechanisms give rise to long-lasting facilitation of phrenic nerve activity, of which phrenic long term facilitation is only one, will be tested. In Aim 2, two of these mechanisms will be investigated, designated the Q and S pathways to phrenic motor facilitation. In the final three aims, the hypotheses will be tested that the Q and S pathways normally suppress one another (Aim 3), but that animals have the capacity to shift between the Q and S pathways phrenic motor facilitation (Aim 4), or that they can engage both pathways at the same time (Aim 5). Such flexibility in achieving facilitation of respiratory nerve activity may impart flexibility as an individual responds to physiological challenges throughout life, such as the onset of disease. A detailed understanding of cellular mechanisms giving rise to phrenic motor facilitation will guide the development of novel therapeutic strategies for severe ventilatory control disorders, including obstructive sleep apnea and respiratory insufficiency in patients with spinal injury or motor neuron disease (ALS). Thus, an important underlying goal of our research is to identify molecules regulating PMF as potential therapeutic targets.